1. Field of the Invention
Chemical analysis of liquids such as water, foodstuffs like milk, and biological liquids is often desirable or necessary. Various elements to facilitate liquid analyses are known. Such elements have often included a reagent for a substance under analysis, termed analyte herein, which reagent, upon contacting a liquid sample containing the analyte, effects formation of a colored material or another detectable change in response to the presence of the analyte. Such elements include, for example, pH test strips and similar indicators wherein a paper or other highly absorbent carrier is impregnated with a material, chemically reactive or otherwise, that responds to contact with liquid containing hydrogen ion or other analyte and either generates color or changes color. Depending on the selection of responsive material, the change is usually qualitative or, at best, semiquantitative. In certain fields, it is often required that analytical techniques yield rapid, quantitative results. Much recent development work has attempted to provide elements useful in diagnostic chemical analysis, where testing of biological liquids including body fluids such as blood, blood serum, urine and the like, must produce highly quantitative results, rapidly and conveniently.
Solution chemical techniques have enjoyed broad acceptance in the clinical laboratory environment, particularly in automated analysis. Such techniques, however, require analyzer equipment often having intricate solution handling and transport capabilities. Analytical equipment of the "wet chemistry" variety, illustrated, for example, in U.S. Pat. No. 2,797,149 can involve complex liquid handling, and may require skilled personnel, both for operation and the precise cleaning that may be needed to avoid sample to sample contamination.
As an alternative to solution chemistry, various multilayer integral elements for non-solution chemical analysis have been proposed. Although essentially dry analysis can offer substantial storage, handling and other conveniences as compared to wet chemistry, variations of the "dry" approach have enjoyed only limited success and have been used primarily for qualitative and semiquantitative test purposes.
2. Description of Related Art
A basic variety of integral elements is described in U.S. Pat. No. 3,092,465. Such multilayer elements use an absorbent fibrous carrier, such as filter paper, impregnated with one or more reagents, typically including a color former, over which is coated a semipermeable membrane. Upon contact with a test liquid, analyte passes through the membrane which prevents passage and absorption into the fibrous carrier of certain interfering components, such as blood cells, that could impair the test result.
Analytical elements that rely on absorbent filter papers or other fibrous media to receive and distribute a liquid sample have not been popular, compared to wet chemical procedures, in applications such as clinical laboratory testing, presumably due to their inability to produce highly accurate, quantitative results. As used herein, the term "fibrous" as applied to materials such as papers and the like refers to materials having discrete fibers, filaments or strands. Exemplary fibers and fiber forms used in preparing analytical elements using various fibrous materials are described, for example, in U.S. Pat. Nos. 3,802,842 and 3,867,258.
It is described in the literature that diagnostic elements using impregnated bibulous materials, such as filter papers, can produce non-uniform test results. In U.S. Pat. No. 3,050,373, it is mentioned that precipitation can occur in the impregnating solutions thereby impairing uniform distribution of reagent within the bibulous matrix. Also, elements using such fibrous, bibulous materials are susceptible to the occurrence of a non-uniform test result phenomenon termed "banding", apparently a deleterious diffusion and chromatographing of reagent chemicals or analyte within the bibulous material.
Integral analytical elements useful in automated test procedures are described in U.S. Pat. Nos. 3,368,872 and 3,526,480. Although, in the 3,526,480 patent, gel matrix materials are discussed as useful for reagent-containing layers, fibrous materials are also described and illustrated. Fibrous materials are also used in reagent layers mentioned in the 3,368,872 patent.
A more recent discussion of an analytical test device using fibrous materials appears in U.S. Pat. No. 3,791,933, which describes a multi-component device for the assay of enzyme substrates and metabolites, such as in body fluids. The device is a clamped array adapted to receive a test sample, filter out or otherwise remove large sample constituents (such as proteins) and effect a test reaction to produce a detectable result, such as the generation of a color. Glass fiber paper is disclosed as assisting in distributing a reaction mixture across a plastic viewing window, preferably porous to minimize lamination problems caused by air entrapment. The glass fiber material apparently merely assists the outward diffusion of liquid sample within that layer, to enlarge the region of the element exhibiting a test result and thereby render the result more easily discernible.
Test devices relying on fibrous matrix materials have exhibited various problems, such as the banding phenomenon referred to previously. As is apparently recognized, the chemical characteristics of the fibrous, bibulous materials (such as absorbent cellulosic filter papers, glass fiber papers, wood, etc) usually proposed as a preferred matrix material for integral analytical elements might impair the accuracy of an analytical result for reasons of chromatographic effects, physical restraint, non-uniform capillary migration or other non-uniform permeation of sample constituents, or for reasons of undesirable chemical binding. Additionally, fibrous materials can generate bias and frustrate highly accurate measurement of an analytical result when the fibrous material is observed by a detection means, due to variations in its properties such as structure, texture and apparent reflectance as seen by a detector for electromagnetic radiation. In the preparation of papers, which appear to be the most popular fibrous matrix material for analytical elements, larger starting fibers are often processed to form smaller tendrils that strengthen the resultant paper.
Various means have been suggested to overcome the banding and other undesirable effects associated with the presence of fibrous matrices in analytical elements. Gelatin and other such materials are described in U.S. Pat. Nos. 3,061,523 and 3,104,209 as useful constituents of impregnating solution for fibrous, bibulous materials, due to the inhibitory effect on banding. However, gelatin and gelatin-like materials in the fibrous, reagent-containing bibulous matrix decrease the rate of sample uptake as compared to a more highly absorbent gelatin-free matrix. Such decreased absorption can leave surface liquid on such test elements and can necessitate washing the element to remove excess sample prior to making a test determination. As a result, upper limits on gelatin concentration have been specified. Such absorption is considered characteristic also of analytical elements using layers solely of gelatin or similar materials, as discussed in U.S. Pat. No. 3,526,480.
In another aspect, it has been suggested, as in U.S. Pat. Nos. 3,368,872 and 3,526,480, that undesirable chromatographic effects can be decreased by immobilization of reagents in an element or by including therein a means to decrease the tendency of spotted or otherwise applied sample to exert a washing effect on incorporated reagent, as by use of certain porous members over an absorbent, reagent-containing material such as fibrous filter paper.
The disturbing effects of banding and the like are referred to also in U.S. Pat. Nos. 3,552,929 and 3,802,842 which propose, respectively, a polymeric coating and meshwork overlayer to be used in connection with fibrous reagent-containing layers in order to minimize such effects.
In pseudo-immersion technique, which can be termed spot confinement, has also been suggested as a means of providing sample-to-sample test result precision, such as between a proteinaceous test liquid and a protein-free calibrator. In accordance with this technique, as is described, for example, in U.S. Pat. Nos. 3,216,804, 3,368,872 and 3,526,480, a barrier is usually included on the element to confine the applied sample, such as a small drop, in a predetermined region of the element's surface. Excess liquid is usually present on the element after sample application. This can create inconvenience in handling if the element is integral and, more seriously, can require precise sample volume delivery when applying sample to the element if test accuracy and precision are to be maintained.
There has been some recognition of the need to promote or avoid, as desired, the migration of reagents and sample constituents, such as between layers of integral analytical elements, for example, as is discussed in U.S. Pat. Nos. 2,761,813; 2,672,431; 2,672,432; 2,677,647; 2,923,669; 3,814,670 and 3,843,452. However, this has generally been in the context of elements for determining the presence of micro-organisms. Such elements generally do not indicate any means to effect or preserve concentrational uniformity, for example, laterally within a layer, and they can require blended layers, the interface of which is characterized by mutual penetration of the adjacent layers.
Until very recently, there has been no effective suggestion in art relating to analytical elements of a layer or other means to receive sample constituents and encourage them to distribute within that means to achieve an apparent concentrational uniformity of analyte, analyte product or other substances that can be metered, in such uniform apparent concentration, to an associated layer for analytical reactions or similar activity. Devices using fibrous materials to provide absorbent layers have sought to overcome the gross effect of such non-uniformity, but they have not succeeded in avoiding the problem. As an example, U.S. Pat. No. 3,715,192 describes an analytical element that provides a hollow space in communication with the surface of a reagent impregnated, preferably fibrous, absorbent capillary material. The hollow space apparently effects more rapid absorption of liquid into the capillary material and minimizes the washing out and chromatographing of reagents, thereby enabling an increased use of reagents that are soluble in liquid under analysis. Also, U.S. Pat. No. 3,723,064 describes an analytical element that includes regions of different effective permeability to an analyte or reaction product of an analyte and produces a plurality of differential, threshold color indications as an analytical result. Although the desirability of a smoothly continuous response is manifest, an element made in accordance with the 3,723,064 patent can only yield an approximate analytical result, the accuracy of which varies inversely with the spacing between thresholds. As the difference in permeability between regions is decreased, in the interest of increased response precision over the intended dynamic range, the complexity of elements made in accordance with the 3,723,064 patent would increase dramatically. Moreover, no suggestion is made as to how one might improve the uniformity and precision of a continuously varying test result and, however, optimized, elements of the 3,723,064 patent would produce a discontinuous response that would apparently be non-uniform within each region of permeability due to non-uniformities associated with the use of filter papers and other fibrous materials.
Improved multilayer integral analytical elements are described in Belgian Pat. No. 801,742. Such elements, preferably formed predominantly from non-fibrous components, can receive a liquid sample and effect distribution of the sample within a spreading layer of the element to obtain a uniform apparent concentration of analyte, other appropriate sample constituent or analyte product and produce uniform, typically quanititative analytical results that, by virtue of their accuracy and precision, can be measured reliably by automated devices, using techniques such as spectrophotometry, fluorometry, etc. Elements disclosed in Belgian Pat. No. 801,742 include spreading layers and reagent layers that contain a reactive or otherwise interactive material that, by virtue of its activity, promotes in the element a radiometrically detectable change, such as a color change.
It has been found that the analytical result obtained using elements of the general type described in Belgian Pat. No. 801,742, adapted to test proteinaceous, aqueous liquids for water-soluble analytes dissolved therein, can be affected by the protein concentration of such liquids. More particularly, it is believed that increased protein concentration can restrain the rate and extent of liquid and analyte transport within the spreading layer and the rate of such transport through the spreading layer. For a given sample volume, more highly proteinaceous analyte-positive liquids produce initially a test result indicating a lower analyte concentration in the sample than would occur at lower protein concentrations and, thereafter, usually produce a test result indicating a higher analyte concentration than would occur at lower protein concentrations. These results are believed to occur due to a restraint in the transport of a sample's solvent and dissolved components within the spreading layer, producing initially in an analyte-positive sample a slower contact of analyte to the indicator composition or other reagent chemistry and producing thereafter a somewhat smaller wetted region for any given sample size. When the indicating reaction is allowed to proceed substantially to completion, a greater amount of analyte is provided to each incremental unit of the sample affected portion of a reagent layer and produces a higher indicated analyte. For accurate determinations, variability in result introduced by protein differences could require that in each instance a protein assay be made to calibrate the analyte assay. Such procedures would be time consuming and add a potential source of error to the analyte determination.
It has been discovered that various surfactant materials can, when included in an effective amount within particularly the spreading layer of elements as described in Belgian Pat. No. 801,742 and in other patents and applications based on the same invention such as U.S. Patent Application Ser. No. 538,072, filed Jan. 2, 1975, inhibit the protein effect discussed above and elsewhere herein.
Surfactants have been described previously in relation to analytical elements. Research Disclosure Publication Volume 126, Item 12626 (October, 1974) refers to analytical elements of the type described in Belgian Pat. No. 801,742 that are intended to analyze liquids for their cholesterol content, using the enzyme cholesterol oxidase. It is described that, in such elements, nonionic surfactants can be used as a coating aid. No concentrations are specified, but about 0.1% is referred to in the 801,742 Belgian patent as useful for such purposes. Also, the publication mentions that it is important that such a surfactant be in the presence of the cholesterol oxidase to assure the complete oxidation of cholesterol. No suggestion is made that the surfactant would be useful in analytical elements for any other purpose or in an analytical element intended for the determination of dissolved analytes. Cholesterol is not dissolved in serum but is transported via associations with lipoproteins. The use of surfactants to disassociate cholesterol esters/protein complexes is also discussed in U.S. Pat. No. 3,907,645.
Surface-active agents are also described in U.S. Pat. No. 3,050,373 as useful for enhancing the density of color produced in a bibulous matrix by glucose detection chemistry using a glucose oxidase, peroxidase and a chromogen. The surfactant is also described as an agent that minimizes the previously discussed banding phenomenon. There is no suggestion in the 3,050,373 patent of having a surfactant in other than the layer of an element containing reagent chemicals and both nonionic and anionic surfactants are discussed as being useful. Also apparently useful for color enhancement purposes are hydrophilic colloids containing a polyvinyl chain, such as polyvinylpyrrolidone and polyvinylalcohol. Wetting agents are also referred to as being useful constituents of reagent layers in U.S. Pat. No. 3,802,842.
The ability to provide improved analytical elements of the type described in Belgian Pat. No. 801,742 to test for analyte dissolved in proteinaceous, aqueous liquids and with minimal differences in test results due to sample-to-sample variations in protein concentration, represents a substantial improvement in the dry chemical analysis of biological liquids.